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Control of radical pair lifetimes by microwave irradiation. Application to photosynthetic reaction centres
3May 1996
CHEMICAL PHYSICS LETTERS
ELSEVIER
Chemical Physics Letters 253 (1996) 361-366
Control of radical pair lifetimes by microwave irradiation. Application to photosynthetic reaction centres Sergei A. Dzuba 1, Ivan I. Proskuryakov 2, Robert J. Hulsebosch, Martin K. Bosch, Peter Gast, Arnold J. Hoff * Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands
Received 11 January 1996; in final form 12 February 1996
Abstract
Radicals produced by illumination or ionizing radiation are often produced in pairs, which quickly decay by recombination or by diffusion and subsequent reactions. For maximizing the yield of products, and for facilitating the study of reaction pathways, it is desirable to minimize the probability of radical pair recombination. We present a way of controlling the radical pair lifetime through the application of a pulse of resonant microwaves in the presence of a magnetic field. Herewith, two radical pair triplet states are coherently populated, from which the pair cannot recombine directly to the singlet ground state because of spin conservation. We illustrate the method with a photosynthetic photochemical reaction, where we have achieved an increase in the radical pair lifetime of up to two orders of magnitude.
1. Introduction Light absorption in reaction centres of photosynthetic bacteria produces within = 200 ps with quantum yield approaching unity the radical pair D+'QA ", where D denotes the primary electron donor, a bacteriochlorophyll dimer, and QA the primary acceptor quinone [1-4]. Under physiological conditions, the electron is subsequently transferred to QR, the sec-
* Corresponding author. Fax +31-71-5275819, E-mail [email protected] ~Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, 630090 Novosibirsk, Russian Federation. 2 Institute of Soil Science and Photosynthesis,Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russian Federation.
ondary acceptor quinone. At cryogenic temperatures this step is blocked [5], and the D+'QA" pair disappears by recombination. The lifetime of D+'QA" radical pairs is = 25 ms at temperatures below 100 K as determined by optical [6-9] and electron paramagnetic resonance (EPR) [6,10] spectroscopy. We show here that the low-temperature lifetime of the D+'QA" pair may be increased by several orders of magnitude when immediately after its creation by a laser flash a brief, intense pulse of resonant microwaves is applied. The spin Hamiltonian for a radical pair in a high magnetic field (here 0.3 T) results in four energy levels corresponding to the spin states [T+ ), Iqba> = cos 4'1S) + sin thlT0), Iqbb) = - - s i n ~ I S ) + cos,;blT 0) and IT_), where T÷, T O and T_ denote three triplet states, S refers to the singlet state and the parameter th, which determines the mixing of T o
000%2614/96/$12.00 © 1996 Elsevier Science B.V. All fights reserved PH S0009-2614(96)00182-0
S.A. Dzuba et a l . / Chemical Physics Letters 253 (1996) 361-366
362
and S states, is a function of the difference in g-values of the two radicals, and their magnetic dipolar and exchange interactions [11]. The radical pair D+'Q -" is created in the singlet state. This state is non-stationary and the system starts to oscillate between the I~a) and [~b) states. Therefore, only these two states are populated initially. IT÷) and IT_ ) may be populated later by transitions from [qbs) and [qbb), for example by spin-lattice relaxation or by applying microwave irradiation at the resonance frequency of these transitions. The Iqba) and I~b) states disappear by recombination to the singlet ground state: D+'QA • --->DQA, as the triplet states of D and QA are energetically inaccessible. Recombination from the IT÷ ) and IT_ ) states is forbidden because of spin conservation. One may therefore expect that when coherently populating these two states with a brief pulse of microwaves resonant between the I~s.b> states and the IT+ ) and IT_ ) states, the radical pair will have a substantially prolonged lifetime. We will show with both pulsed EPR and optical absorption spectroscopy that an
increase in the D+'QA" lifetime by a factor of 100 to up to 3 s at 7.5 K may thus be achieved.
2. Experimental Reaction
centres
of the
purple
bacterium
Rhodobacter (Rb.) sphaeroides R26, in which the native paramagnetic iron was replaced by the diamagnetic Zn 2÷ ion were prepared as in Ref. [12]. A typical EPR sample contained 60-70% (v/v) glycerol as cryoprotectant. The final reaction centre concentration was about 50 /zM. The samples were frozen in the dark at 77 K and stored until use at this temperature. Pulsed EPR measurements were carried out with a home-built pulsed EPR spectrometer [13]. Two types of pulse sequence were employed (inset Fig. 1). A two-pulse electron spin echo sequence (pulse1 and pulse2, both of 16 ns duration), applied at the resonance frequency of electron spin transitions in the magnetic field ( = 0.3 mT), monitored the concentra-
800 t
I
":'. --., >'
" ._ o
to
T=I 5 K
600
LI T I T I P1 P2 8 c h o
laser
400
laser
.
.
.
.
.
.
echo
200
0
......................
-200
, 0.0
0.2
3 - p , l-s e 0.4
,
,
0.6
0.8
1.0
t (s) Fig. 1. The electron spin echo signal as a function of the time delay t after the laser flash of the radical pair D+QA" in photosynthetic reaction centres of the purple bacterium Rhodobacter sphaeroides R26, in which the native paramagnetic iron was replaced by the diamagnetic Zn 2+ ion. Two pulse sequences were utilized, as shown in the inset. In the two-pulse scheme a laser flash at 890 nm of 5 ns duration after a variable delay t was followed by two echo-forming microwave pulses (pulsel and pulse2, both of 16 ns duration). The time interval r between the two pulses (200 ns) is much shorter than the typical delay t after the laser flash. The negative component at longer delay times is attributed to spin-lattice relaxation, which populates the non-reactive IT± ) states. In the three-pulse experiment, an additional 8 ns microwave pulseO is applied 1 /zs after the laser flash. This pulse rotates the magnetisation vector and therefore transfers the IDa) and IO b) states to the IT+ ) and ~ l ) states much more efficiently than the spin-lattice relaxation. This gives rise to a much stronger negative component than that obtained in the two-pulse experiment.
S.A. Dzuba et al./ Chemical Physics Letters 253 (1996) 361-366
tion of the radical pair at a variable delay time t after a laser flash at 890 nm of 5 ns duration, provided by a Continuum Surelite I laser pumping an optical parametric oscillator (output power -- 300 mJ/pulse). The amplitudes and relative durations of the pulses were adjusted to provide maximum echo amplitude. (Note that for spin-polarized radical pair EPR signals, the maximum echo amplitude is obtained for rotation angles that are somewhat different from the usual 90-180 ° sequence [14].) The time interval between the two pulses (200 ns) was much shorter than the typical delay t after the laser flash. In the three-pulse experiment, an additional 8 ns microwave pulseO was applied 1 /xs after the laser flash. This pulse rotates the magnetisation vector, transferring coherently half the population of the ICl)a) and [qbb) states to the IT+) and IT_) states. Subsequently, a two-pulse sequence was applied as above.
3. Results and discussion
Fig. 1 shows the amplitude of the electron spin echo signal after a two- and three-pulse sequence as a function of the time delay t after the laser flash in Zn 2+-reconstituted reaction centres of Rb.
2
250
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-; ,i .,,.. ~'
j , ~
o=o 125
¢n t-
-I
0.5
1.0
1.5
2.0
log(T)
25 K
0
-125 -250 0.0
' 0.2
' 0.4
'
'
0.6
0.8
363
sphaeroides R26. The amplitude of the echo signal is proportional to the difference between the populations of the IT_+) and [dPa.b) states. Initially, the radical pair is formed in a singlet state. Therefore only n a and n b are non-zero at small t. The singlet state disappears by recombination, leading to n~, n b ~ 0 at large t. This process is represented by the fast initial decay of the signal, with a lifetime = 25 ms. At long delays a small, slow-decaying negative signal develops, which we ascribe to population of the IT_+) states through spin-lattice relaxation between the [(I)a.b) and IT+ ) states during the lifetime of D÷'QA ". This process leads to sublevel population inversion at long delays compared to the initial population, because IT+) and IT_ ) are non-reactive in the magnetic field. It follows that at larger t a weak negative echo is detected, which decays by transition from the triplet to the reactive Iqba) and [dPb) states, again through spin-lattice relaxation 3. The long-living negative component of the echo decay kinetics becomes remarkably stronger (about 20% of the total signal at 15 K) in a three-pulse experiment, where an additional 8 ns microwave pulseO is applied 1 /zs after the laser flash (Fig. 1). We attribute the strong enhancement of the long-lived negative echo signal to the transfer of the I~a) and [@b) sublevel populations to the isolated IT+ ) and IT_ ) sublevels by pulseO. This pulse rotates the magnetisation vector and therefore transfers coherently the population of the I~a) and [~b) states to the IT+ ) and IT_ ) states much more efficiently than spin-lattice relaxation. As the sublevel populations are inverted with respect to those directly after the flash, the long-lived echo has a negative sign. The (mono-exponential) decay rate k of the longliving negative component of the three-pulse echo decay kinetics is strongly temperature dependent (Fig. 2), whereas the rate constant of the fast-decaying positive signal is practically temperature indepen-
1.0
t (s) Fig. 2. Temperature dependence o f the spin echo decay as in Fig. 1, measured with the three-pulse sequence. The rate constant o f the fast-decaying positive signal is determined by recombination o f the radical pair, and is practically temperature independent. The temperature dependence o f the rate constant k o f the mono-exponential, slowly decaying negative component is shown in the inset as a log-log plot o f k vs. T. The slope is 2.8+0.2, corresponding to k ot T 2-s.
3 The amplitude of the echo signal is the result of summing the absorptive and emissive contributions of the four transitions between the radical pair states. Because the magnetic couplings between the two radicals and their g-value difference is small compared to the linewidth and the spectral range covered by the microwave field, considerable cancellation effects occur, and the echo intensity is only a few percent of the intensity expected for a fully polarized (absorptive/emissive) radical EPR transition.
364
S.A. Dzuba et al.// Chemical Physics Letters 253 (1996) 361-366
332
334
a (roT) o o
~ m
x3~i 0.0
|
i
I
0.5
1.0
1.5
2.0
r (~s) Fig. 3. Electron spin echo envelope modulation at T = 15 K obtained for the signal immediately after the laser flash with a two-pulse sequence (solid line) and for the signal at t = 150 ms, with pulseO of the three-pulse sequence enhancing the population of the long-lived triplet states (dotted line). The inset shows the field dependence of both signals. For comparison the baseline recorded in the absence of the laser flash is also given (dashed line). The modulation patterns demonstrate that the nature of the radicals giving rise to the two echo signals is the same, the different sign of the signal recorded at a delay of 150 ms being due to inversion of the sublevel populations.
dent, agreeing with the known temperature dependence of recombination of the radical pair D÷'QA" [6-10]. The inset of Fig. 2 shows a log-log plot of k vs. T. The slope is 2.8 ___0.2, corresponding to k ct T 2"8, indicating that the decay is mainly governed by a phonon-bottleneck spin-lattice relaxation process [15]. The temperature dependence of the decay rate of the long-living negative component of the twopulse echo decay kinetics (Fig. 1) is identical to that of the three-pulse echo (data not shown). This strongly supports our assignment of this component to the decay of the ~T± ) population generated through spin-lattice relaxation between the I~a,b) and IT_+) states during the lifetime of D+'QA ". To confirm that the short- and long-lived echo signals are both due to the same radical species, we performed an electron spin echo envelope modulation experiment [16], in which the echo signal is detected as a function of the delay time ~-. The resulting modulation is a fingerprint of the radicals under study. Fig. 3 shows that the echo modulation for the long-lived echo signal at t = 150 ms, with pulseO of the three-pulse sequence enhancing the population of the long-lived triplet states, is within
experimental error the same as that obtained immediately after the laser flash with a two-pulse sequence. (In this particular case, the modulation is induced by dipolar and exchange interactions in the radical pair [12].) Fig. 3 (inset) also presents data on the field dependence of the echo signal at short and long delays after the laser flash. The two spectra are (apart from the sign) similar, evidencing that the nature of the radical species does not change with time. Note that the shape of the spectra in the inset of Fig. 3 is quite different from that of the polarized EPR spectrum of the D+'QA" radical pair [11]. This is caused by the large amplitude of the microwave field in the spin echo experiments, which spans a considerable part of the spectral width, smearing out the details of the polarized EPR spectrum obtained with low-intensity microwaves. The total polarization of the spectrum does not integrate to zero, as it does (approximately) for the polarized EPR spectrum. This incomplete cancellation of the absorptive and emissive parts of the field-swept electron spin echo spectrum is probably caused by the combination of random spatial distribution and an excitation width comparable to the spectral width. For further confirmation, we have monitored inside the microwave cavity of the EPR spectrometer the recovery of photobleaching at 890 nm, where D absorbs. The sample was excited by a 5 ns laser flash at 590 nm and an 8 ns microwave pulse was applied 1 /xs after the flash, similar to pulseO in the spin echo experiment. When the magnetic field was outside the region for radical pair EPR (about 5 mT off-resonance), the usual decay kinetics of the optical transmission were observed, as the weak long-lived component observable with spin echo under such conditions was below the noise level 4. Within the
4 In the optical kinetic experiment the signals from all radical pairs add, while in the electron spin echo monitored kinetics, the signal is the result of large contributions of opposite sign (the absorptive and emissive transitions between the radical pair states), and constitutes only a few percent of the signal that would be obtained for a fully polarized transition. Hence, for the spin echo signal, small differences in the cancellation produced by thermal relaxation to the IT± ) levels will have a comparatively large effect on the signal amplitude. For the optical signal, the slow component due to the same minor degree of IT± ) population is hardly observable.
S.A. Dzuba et al./ Chemical Physics Letters 253 (1996) 361-366
tO
Ck
o i
I
i
i
0.00
0.05
0.10
0.15
time
(s)
Fig. 4. The decay of the radical pair D+'Q~." monitored via the changes in light transmission at 890 nm, where D absorbs. Optical measurements were performed inside the EPR cavity using a flat quartz cell. The sample was excited by a 5 ns laser flash at 590 nm. An 8 ns microwave pulse was applied 1 /xs after the flash, similar to pulseO in the spin echo experiment. The figure displays the difference between the on-resonance and off-resonance optical decay curves taken at 25 K. The risetime of the curve corresponds to the lifetime of the singlet radical pair. The rate constant of the decay (13 s - I ) agrees well with the decay rate constant of the spin echo detected slow component at the same temperature (14 s - I , Fig. 2).
resonance field region, however, an additional longlived component became apparent, which is attributed to enhanced population of the non-reactive triplet sublevels. Fig. 4 displays the difference between the on-resonance and off-resonance optical decay curves taken at 25 K. The fast rise of the curve corresponds to the normal recombination kinetics of the singlet D+'Q;. • radical pair. The rate constant of the slow decay (13 s - I ) agrees well with the decay rate constant of the spin echo detected slow component at the same temperature (14 s - l , cf. Fig. 2). While, to the best of our knowledge, the present method of prolonging the radical pair lifetime by coherently transferring population from the mixed ](IDa) and [~b) levels to the IT+ levels has not been previously applied 5, it bears a certain similarity to earlier work in which the effect of microwave irradi-
5 After completion of this manuscript, we leamed that microwave-induced transfer to the IT± > states was suggested in K.M. Salikhov and Yu.N. Molin, J. Phys. Chem. 97 (1993) 13259.
365
ation on chemical reactions was studied. For example, in so-called reaction-yield detected magnetic resonance (RYDMR) the effect of resonant microwaves on the product yield is monitored, either through measuring the recombinational luminescence (reviewed in Ref. [17]), or by monitoring the singlet ground-state absorbance of one of the precursors [18-20]. In these experiments, however, the effects of microwave irradiation on radical pair lifetimes are minor. In another form of RYDMR, labelled product-yield ESR, the effect of microwaves on the product yield from a triplet radical pair is monitored through spin trapping and ESR spectroscopy ([21] and references therein). Here, the radical pair lifetime is actually decreased by (non-coherently) transferring population from the IT.z_ - ) states to the mixed IS), IT0) states. None of the earlier work has achieved the considerable enhancement of radical pair lifetime reported here. We have demonstrated the above method for prolonging the radical pair lifetime with a photosynthetic photochemical reaction, but it should be generally applicable for radical pairs produced by light or ionizing radiation in vivo and in vitro. While the present experiments have been carried out at low temperature, it suffices that spin relaxation is slower than radical recombination, a situation that is generally obtained also at higher temperatures in liquid solution. It should be noted that our pulseO is quite short, 8 ns, shorter than or comparable to the primary radical pair recombination times generally encountered in liquid solution. In principle, the pulse can be delivered concomitant with the laser pulse, thus converting at least a sizeable fraction of the primary radical pairs to the non-reactive IT_+) states. While in liquid solution the IT_+) levels may also become populated through radical re-encounters or, when the exchange interaction between the radicals remains large for an appreciable time (bringing either the IT_ ) or the IT+ > level close to the singlet level), through the non-secular part of the hyperfine interaction (reviewed in Ref. [22]), we believe that in most cases our method for coherently populating the IT_+> states of the initial radical pair will be much more efficient. We note that for prolonging the radical pair lifetime we need only to populate the IT+ > and IT_ > triplet sublevels and kinetically isolate them by an
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external magnetic field. In zero external magnetic field all four D÷'Q~, • levels are more or less equally populated, as their splittings are small and they all contain at least some singlet character. When switching on a magnetic field immediately after the laser flash, the IT± ) levels will be isolated, and a situation is obtained similar to that after our pulseO, with again half the radical pairs in the long-living IT+_) states. Experiments to verify this effect are in progress.
[6] [7] [8] [9] [10] [11]
Acknowledgements We thank Mr. A.H.M. de Wit for growing the bacteria, and Ms. S.J. Jansen for help with preparing the reaction centres. SAD and IIP acknowledge travel grants from INTAS and NWO (Netherlands Organization for Scientific Research).
References
[12] [13] [14] [15] [16]
[17] [18]
[1] G. Feher and M.Y. Okamura, in: The photosynthetic bacteria, eds. R.K. Clayton and W.R. Sistrom (Plenum Press, New York, 1978) p. 349. [2] W.W. Parson in: Chlorophylls, ed. H. Scheer (CRC Press, Boca Raton, FL, 1991) p. 1153. [3] C. Kirmaier and D. Holten, in: The photosynthetic reaction center, eds. J. Deisenhofer and J.R. Norris, Vol. II (Academic Press, San Diego, CA, 1993) p. 49. [4] K. Dressier, E. Umlauf, S. Schmidt, P. Hamm, W. Zinth, S. Buchanan and H. Michel, Chem. Phys. Letters 183 (1991) 270. [5] W.W. Parson, in: The photosynthetic bacteria, eds. R.K.
[ 19] [20]
[21] [22]
Clayton and W.R. Sistrom (Plenum Press, New York, 1978) p. 455. .I.D. McElroy, D.C. Mauzerall and G. Feher, Biochim. Biophys. Acta 333 (1974) 261. D. Kleinfield, M.Y. Okamura and G. Feher, Biochemistry 23 (1984) 5780. P. Parot, J. Thiery and A. Verm6glio, Biochim. Biophys. Acta 893 (1987) 534. R.J. Debus, G. Feher and M.Y. Okamura, Biochemistry 25 (1986) 2276. J.S. van den Brink, R.J, Hulsebosch, P. Gast, P.J. Hore and A.J. Hoff, Biochemistry 33 (1994) 13668. P.J. Hore, in: Advanced EPR, applications in biology and biochemistry, ed. A.J. Hoff (Elsevier, Amsterdam, 1989) p. 405. S.A. Dzuba, P. Gast and A.J. Hoff, Chem. Phys. Letters 236 (1995) 595. M.K. Bosch, PhD Thesis, Leiden University, 1995. J. Tang, M.C. Thumauer and J.R. Norris, Chem. Phys. Letters 219 (1994) 283. C.P. Poole and H.A. Farach, Relaxation in magnetic resonance (Academic Press, New York, 1971). S.A. Dikanov and Yu.D. Tsvetkov, Electron spin echo modulation (ESEEM) spectroscopy (CRC Press, Boca Raton, FL, 1992). E.L. Frankevich and A.I. Pristupa, in: Triplet state ODMR spectroscopy, ed. R. Clarke (Wiley, New York, 1982) p. 137. M.K. Bowman, D.E. Budil, G.L. Closs, C.A. Kostka, C.A. Wraight and J.R. Norris, Proc. Natl, Acad. Sci. USA 78 (1981) 3305. K.W. Mochl, E.J. Lous and A.J. Hoff, Chem. Phys. Letters 121 (1985) 22. W. Lersch and M.E. Michel-Beyerle, in: Advanced EPR, applications in biology and biochemistry, ed. A.J. Hoff (Elsevier, Amsterdam, 1989) p. 685. N.E. Polyakov, Y. Konishi, M. Okazaki and K. Toriyama, J. Phys. Chem. 98 (1994) 10558. K.A. McLauchlan, in: Advanced EPR, applications in biology and biochemistry, ed. A.J. Hoff (Elsevier, Amsterdam, 1989) p. 345.
CHEMICAL PHYSICS LETTERS
ELSEVIER
Chemical Physics Letters 253 (1996) 361-366
Control of radical pair lifetimes by microwave irradiation. Application to photosynthetic reaction centres Sergei A. Dzuba 1, Ivan I. Proskuryakov 2, Robert J. Hulsebosch, Martin K. Bosch, Peter Gast, Arnold J. Hoff * Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA Leiden, The Netherlands
Received 11 January 1996; in final form 12 February 1996
Abstract
Radicals produced by illumination or ionizing radiation are often produced in pairs, which quickly decay by recombination or by diffusion and subsequent reactions. For maximizing the yield of products, and for facilitating the study of reaction pathways, it is desirable to minimize the probability of radical pair recombination. We present a way of controlling the radical pair lifetime through the application of a pulse of resonant microwaves in the presence of a magnetic field. Herewith, two radical pair triplet states are coherently populated, from which the pair cannot recombine directly to the singlet ground state because of spin conservation. We illustrate the method with a photosynthetic photochemical reaction, where we have achieved an increase in the radical pair lifetime of up to two orders of magnitude.
1. Introduction Light absorption in reaction centres of photosynthetic bacteria produces within = 200 ps with quantum yield approaching unity the radical pair D+'QA ", where D denotes the primary electron donor, a bacteriochlorophyll dimer, and QA the primary acceptor quinone [1-4]. Under physiological conditions, the electron is subsequently transferred to QR, the sec-
* Corresponding author. Fax +31-71-5275819, E-mail [email protected] ~Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, 630090 Novosibirsk, Russian Federation. 2 Institute of Soil Science and Photosynthesis,Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russian Federation.
ondary acceptor quinone. At cryogenic temperatures this step is blocked [5], and the D+'QA" pair disappears by recombination. The lifetime of D+'QA" radical pairs is = 25 ms at temperatures below 100 K as determined by optical [6-9] and electron paramagnetic resonance (EPR) [6,10] spectroscopy. We show here that the low-temperature lifetime of the D+'QA" pair may be increased by several orders of magnitude when immediately after its creation by a laser flash a brief, intense pulse of resonant microwaves is applied. The spin Hamiltonian for a radical pair in a high magnetic field (here 0.3 T) results in four energy levels corresponding to the spin states [T+ ), Iqba> = cos 4'1S) + sin thlT0), Iqbb) = - - s i n ~ I S ) + cos,;blT 0) and IT_), where T÷, T O and T_ denote three triplet states, S refers to the singlet state and the parameter th, which determines the mixing of T o
000%2614/96/$12.00 © 1996 Elsevier Science B.V. All fights reserved PH S0009-2614(96)00182-0
S.A. Dzuba et a l . / Chemical Physics Letters 253 (1996) 361-366
362
and S states, is a function of the difference in g-values of the two radicals, and their magnetic dipolar and exchange interactions [11]. The radical pair D+'Q -" is created in the singlet state. This state is non-stationary and the system starts to oscillate between the I~a) and [~b) states. Therefore, only these two states are populated initially. IT÷) and IT_ ) may be populated later by transitions from [qbs) and [qbb), for example by spin-lattice relaxation or by applying microwave irradiation at the resonance frequency of these transitions. The Iqba) and I~b) states disappear by recombination to the singlet ground state: D+'QA • --->DQA, as the triplet states of D and QA are energetically inaccessible. Recombination from the IT÷ ) and IT_ ) states is forbidden because of spin conservation. One may therefore expect that when coherently populating these two states with a brief pulse of microwaves resonant between the I~s.b> states and the IT+ ) and IT_ ) states, the radical pair will have a substantially prolonged lifetime. We will show with both pulsed EPR and optical absorption spectroscopy that an
increase in the D+'QA" lifetime by a factor of 100 to up to 3 s at 7.5 K may thus be achieved.
2. Experimental Reaction
centres
of the
purple
bacterium
Rhodobacter (Rb.) sphaeroides R26, in which the native paramagnetic iron was replaced by the diamagnetic Zn 2÷ ion were prepared as in Ref. [12]. A typical EPR sample contained 60-70% (v/v) glycerol as cryoprotectant. The final reaction centre concentration was about 50 /zM. The samples were frozen in the dark at 77 K and stored until use at this temperature. Pulsed EPR measurements were carried out with a home-built pulsed EPR spectrometer [13]. Two types of pulse sequence were employed (inset Fig. 1). A two-pulse electron spin echo sequence (pulse1 and pulse2, both of 16 ns duration), applied at the resonance frequency of electron spin transitions in the magnetic field ( = 0.3 mT), monitored the concentra-
800 t
I
":'. --., >'
" ._ o
to
T=I 5 K
600
LI T I T I P1 P2 8 c h o
laser
400
laser
.
.
.
.
.
.
echo
200
0
......................
-200
, 0.0
0.2
3 - p , l-s e 0.4
,
,
0.6
0.8
1.0
t (s) Fig. 1. The electron spin echo signal as a function of the time delay t after the laser flash of the radical pair D+QA" in photosynthetic reaction centres of the purple bacterium Rhodobacter sphaeroides R26, in which the native paramagnetic iron was replaced by the diamagnetic Zn 2+ ion. Two pulse sequences were utilized, as shown in the inset. In the two-pulse scheme a laser flash at 890 nm of 5 ns duration after a variable delay t was followed by two echo-forming microwave pulses (pulsel and pulse2, both of 16 ns duration). The time interval r between the two pulses (200 ns) is much shorter than the typical delay t after the laser flash. The negative component at longer delay times is attributed to spin-lattice relaxation, which populates the non-reactive IT± ) states. In the three-pulse experiment, an additional 8 ns microwave pulseO is applied 1 /zs after the laser flash. This pulse rotates the magnetisation vector and therefore transfers the IDa) and IO b) states to the IT+ ) and ~ l ) states much more efficiently than the spin-lattice relaxation. This gives rise to a much stronger negative component than that obtained in the two-pulse experiment.
S.A. Dzuba et al./ Chemical Physics Letters 253 (1996) 361-366
tion of the radical pair at a variable delay time t after a laser flash at 890 nm of 5 ns duration, provided by a Continuum Surelite I laser pumping an optical parametric oscillator (output power -- 300 mJ/pulse). The amplitudes and relative durations of the pulses were adjusted to provide maximum echo amplitude. (Note that for spin-polarized radical pair EPR signals, the maximum echo amplitude is obtained for rotation angles that are somewhat different from the usual 90-180 ° sequence [14].) The time interval between the two pulses (200 ns) was much shorter than the typical delay t after the laser flash. In the three-pulse experiment, an additional 8 ns microwave pulseO was applied 1 /xs after the laser flash. This pulse rotates the magnetisation vector, transferring coherently half the population of the ICl)a) and [qbb) states to the IT+) and IT_) states. Subsequently, a two-pulse sequence was applied as above.
3. Results and discussion
Fig. 1 shows the amplitude of the electron spin echo signal after a two- and three-pulse sequence as a function of the time delay t after the laser flash in Zn 2+-reconstituted reaction centres of Rb.
2
250
~" 1
-; ,i .,,.. ~'
j , ~
o=o 125
¢n t-
-I
0.5
1.0
1.5
2.0
log(T)
25 K
0
-125 -250 0.0
' 0.2
' 0.4
'
'
0.6
0.8
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sphaeroides R26. The amplitude of the echo signal is proportional to the difference between the populations of the IT_+) and [dPa.b) states. Initially, the radical pair is formed in a singlet state. Therefore only n a and n b are non-zero at small t. The singlet state disappears by recombination, leading to n~, n b ~ 0 at large t. This process is represented by the fast initial decay of the signal, with a lifetime = 25 ms. At long delays a small, slow-decaying negative signal develops, which we ascribe to population of the IT_+) states through spin-lattice relaxation between the [(I)a.b) and IT+ ) states during the lifetime of D÷'QA ". This process leads to sublevel population inversion at long delays compared to the initial population, because IT+) and IT_ ) are non-reactive in the magnetic field. It follows that at larger t a weak negative echo is detected, which decays by transition from the triplet to the reactive Iqba) and [dPb) states, again through spin-lattice relaxation 3. The long-living negative component of the echo decay kinetics becomes remarkably stronger (about 20% of the total signal at 15 K) in a three-pulse experiment, where an additional 8 ns microwave pulseO is applied 1 /zs after the laser flash (Fig. 1). We attribute the strong enhancement of the long-lived negative echo signal to the transfer of the I~a) and [@b) sublevel populations to the isolated IT+ ) and IT_ ) sublevels by pulseO. This pulse rotates the magnetisation vector and therefore transfers coherently the population of the I~a) and [~b) states to the IT+ ) and IT_ ) states much more efficiently than spin-lattice relaxation. As the sublevel populations are inverted with respect to those directly after the flash, the long-lived echo has a negative sign. The (mono-exponential) decay rate k of the longliving negative component of the three-pulse echo decay kinetics is strongly temperature dependent (Fig. 2), whereas the rate constant of the fast-decaying positive signal is practically temperature indepen-
1.0
t (s) Fig. 2. Temperature dependence o f the spin echo decay as in Fig. 1, measured with the three-pulse sequence. The rate constant o f the fast-decaying positive signal is determined by recombination o f the radical pair, and is practically temperature independent. The temperature dependence o f the rate constant k o f the mono-exponential, slowly decaying negative component is shown in the inset as a log-log plot o f k vs. T. The slope is 2.8+0.2, corresponding to k ot T 2-s.
3 The amplitude of the echo signal is the result of summing the absorptive and emissive contributions of the four transitions between the radical pair states. Because the magnetic couplings between the two radicals and their g-value difference is small compared to the linewidth and the spectral range covered by the microwave field, considerable cancellation effects occur, and the echo intensity is only a few percent of the intensity expected for a fully polarized (absorptive/emissive) radical EPR transition.
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S.A. Dzuba et al.// Chemical Physics Letters 253 (1996) 361-366
332
334
a (roT) o o
~ m
x3~i 0.0
|
i
I
0.5
1.0
1.5
2.0
r (~s) Fig. 3. Electron spin echo envelope modulation at T = 15 K obtained for the signal immediately after the laser flash with a two-pulse sequence (solid line) and for the signal at t = 150 ms, with pulseO of the three-pulse sequence enhancing the population of the long-lived triplet states (dotted line). The inset shows the field dependence of both signals. For comparison the baseline recorded in the absence of the laser flash is also given (dashed line). The modulation patterns demonstrate that the nature of the radicals giving rise to the two echo signals is the same, the different sign of the signal recorded at a delay of 150 ms being due to inversion of the sublevel populations.
dent, agreeing with the known temperature dependence of recombination of the radical pair D÷'QA" [6-10]. The inset of Fig. 2 shows a log-log plot of k vs. T. The slope is 2.8 ___0.2, corresponding to k ct T 2"8, indicating that the decay is mainly governed by a phonon-bottleneck spin-lattice relaxation process [15]. The temperature dependence of the decay rate of the long-living negative component of the twopulse echo decay kinetics (Fig. 1) is identical to that of the three-pulse echo (data not shown). This strongly supports our assignment of this component to the decay of the ~T± ) population generated through spin-lattice relaxation between the I~a,b) and IT_+) states during the lifetime of D+'QA ". To confirm that the short- and long-lived echo signals are both due to the same radical species, we performed an electron spin echo envelope modulation experiment [16], in which the echo signal is detected as a function of the delay time ~-. The resulting modulation is a fingerprint of the radicals under study. Fig. 3 shows that the echo modulation for the long-lived echo signal at t = 150 ms, with pulseO of the three-pulse sequence enhancing the population of the long-lived triplet states, is within
experimental error the same as that obtained immediately after the laser flash with a two-pulse sequence. (In this particular case, the modulation is induced by dipolar and exchange interactions in the radical pair [12].) Fig. 3 (inset) also presents data on the field dependence of the echo signal at short and long delays after the laser flash. The two spectra are (apart from the sign) similar, evidencing that the nature of the radical species does not change with time. Note that the shape of the spectra in the inset of Fig. 3 is quite different from that of the polarized EPR spectrum of the D+'QA" radical pair [11]. This is caused by the large amplitude of the microwave field in the spin echo experiments, which spans a considerable part of the spectral width, smearing out the details of the polarized EPR spectrum obtained with low-intensity microwaves. The total polarization of the spectrum does not integrate to zero, as it does (approximately) for the polarized EPR spectrum. This incomplete cancellation of the absorptive and emissive parts of the field-swept electron spin echo spectrum is probably caused by the combination of random spatial distribution and an excitation width comparable to the spectral width. For further confirmation, we have monitored inside the microwave cavity of the EPR spectrometer the recovery of photobleaching at 890 nm, where D absorbs. The sample was excited by a 5 ns laser flash at 590 nm and an 8 ns microwave pulse was applied 1 /xs after the flash, similar to pulseO in the spin echo experiment. When the magnetic field was outside the region for radical pair EPR (about 5 mT off-resonance), the usual decay kinetics of the optical transmission were observed, as the weak long-lived component observable with spin echo under such conditions was below the noise level 4. Within the
4 In the optical kinetic experiment the signals from all radical pairs add, while in the electron spin echo monitored kinetics, the signal is the result of large contributions of opposite sign (the absorptive and emissive transitions between the radical pair states), and constitutes only a few percent of the signal that would be obtained for a fully polarized transition. Hence, for the spin echo signal, small differences in the cancellation produced by thermal relaxation to the IT± ) levels will have a comparatively large effect on the signal amplitude. For the optical signal, the slow component due to the same minor degree of IT± ) population is hardly observable.
S.A. Dzuba et al./ Chemical Physics Letters 253 (1996) 361-366
tO
Ck
o i
I
i
i
0.00
0.05
0.10
0.15
time
(s)
Fig. 4. The decay of the radical pair D+'Q~." monitored via the changes in light transmission at 890 nm, where D absorbs. Optical measurements were performed inside the EPR cavity using a flat quartz cell. The sample was excited by a 5 ns laser flash at 590 nm. An 8 ns microwave pulse was applied 1 /xs after the flash, similar to pulseO in the spin echo experiment. The figure displays the difference between the on-resonance and off-resonance optical decay curves taken at 25 K. The risetime of the curve corresponds to the lifetime of the singlet radical pair. The rate constant of the decay (13 s - I ) agrees well with the decay rate constant of the spin echo detected slow component at the same temperature (14 s - I , Fig. 2).
resonance field region, however, an additional longlived component became apparent, which is attributed to enhanced population of the non-reactive triplet sublevels. Fig. 4 displays the difference between the on-resonance and off-resonance optical decay curves taken at 25 K. The fast rise of the curve corresponds to the normal recombination kinetics of the singlet D+'Q;. • radical pair. The rate constant of the slow decay (13 s - I ) agrees well with the decay rate constant of the spin echo detected slow component at the same temperature (14 s - l , cf. Fig. 2). While, to the best of our knowledge, the present method of prolonging the radical pair lifetime by coherently transferring population from the mixed ](IDa) and [~b) levels to the IT+ levels has not been previously applied 5, it bears a certain similarity to earlier work in which the effect of microwave irradi-
5 After completion of this manuscript, we leamed that microwave-induced transfer to the IT± > states was suggested in K.M. Salikhov and Yu.N. Molin, J. Phys. Chem. 97 (1993) 13259.
365
ation on chemical reactions was studied. For example, in so-called reaction-yield detected magnetic resonance (RYDMR) the effect of resonant microwaves on the product yield is monitored, either through measuring the recombinational luminescence (reviewed in Ref. [17]), or by monitoring the singlet ground-state absorbance of one of the precursors [18-20]. In these experiments, however, the effects of microwave irradiation on radical pair lifetimes are minor. In another form of RYDMR, labelled product-yield ESR, the effect of microwaves on the product yield from a triplet radical pair is monitored through spin trapping and ESR spectroscopy ([21] and references therein). Here, the radical pair lifetime is actually decreased by (non-coherently) transferring population from the IT.z_ - ) states to the mixed IS), IT0) states. None of the earlier work has achieved the considerable enhancement of radical pair lifetime reported here. We have demonstrated the above method for prolonging the radical pair lifetime with a photosynthetic photochemical reaction, but it should be generally applicable for radical pairs produced by light or ionizing radiation in vivo and in vitro. While the present experiments have been carried out at low temperature, it suffices that spin relaxation is slower than radical recombination, a situation that is generally obtained also at higher temperatures in liquid solution. It should be noted that our pulseO is quite short, 8 ns, shorter than or comparable to the primary radical pair recombination times generally encountered in liquid solution. In principle, the pulse can be delivered concomitant with the laser pulse, thus converting at least a sizeable fraction of the primary radical pairs to the non-reactive IT_+) states. While in liquid solution the IT_+) levels may also become populated through radical re-encounters or, when the exchange interaction between the radicals remains large for an appreciable time (bringing either the IT_ ) or the IT+ > level close to the singlet level), through the non-secular part of the hyperfine interaction (reviewed in Ref. [22]), we believe that in most cases our method for coherently populating the IT_+> states of the initial radical pair will be much more efficient. We note that for prolonging the radical pair lifetime we need only to populate the IT+ > and IT_ > triplet sublevels and kinetically isolate them by an
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external magnetic field. In zero external magnetic field all four D÷'Q~, • levels are more or less equally populated, as their splittings are small and they all contain at least some singlet character. When switching on a magnetic field immediately after the laser flash, the IT± ) levels will be isolated, and a situation is obtained similar to that after our pulseO, with again half the radical pairs in the long-living IT+_) states. Experiments to verify this effect are in progress.
[6] [7] [8] [9] [10] [11]
Acknowledgements We thank Mr. A.H.M. de Wit for growing the bacteria, and Ms. S.J. Jansen for help with preparing the reaction centres. SAD and IIP acknowledge travel grants from INTAS and NWO (Netherlands Organization for Scientific Research).
References
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